Active Thiol Research Articles

Hydrogen Sulfide Mediates Tumor Cell Resistance to Thioredoxin Inhibitor.

Thioredoxin (Trx) is a pro-oncogenic molecule that underlies tumor initiation, progression and chemo-resistance. PX-12, a Trx inhibitor, has been used to treat certain tumors. Currently, factors predicting tumor sensitivity to PX-12 are unclear. Given that hydrogen sulfide (H2S), a gaseous bio-mediator, promotes Trx activity, we speculated that it might affect tumor response to PX-12. Here, we tested this possibility. Exposure of several different types of tumor cells to PX-12 caused cell death, which was reversely correlated with the levels of H2S-synthesizing enzyme CSE and endogenous H2S. Inhibition of CSE sensitized tumor cells to PX-12, whereas addition of exogenous H2S elevated PX-12 resistance. Further experiments showed that H2S abolished PX-12-mediated inhibition on Trx. Mechanistic analyses revealed that H2S stimulated Trx activity. It promoted Trx from the oxidized to the reduced state. In addition, H2S directly cleaved the disulfide bond in PX-12, causing PX-12 deactivation. Additional studies found that, besides Trx, PX-12 also interacted with the thiol residues of other proteins. Intriguingly, H2S-mediated cell resistance to PX-12 could also be achieved through promotion of the thiol activity of these proteins. Addition of H2S-modified protein into culture significantly enhanced cell resistance to PX-12, whereas blockade of extracellular sulfhydryl residues sensitized cells to PX-12. Collectively, our study revealed that H2S mediated tumor cell resistance to PX-12 through multiple mechanisms involving induction of thiol activity in multiple proteins and direct inactivation of PX-12. H2S could be used to predict tumor response to PX-12 and could be targeted to enhance the therapeutic efficacy of PX-12.

Cu2+-mediated Fluorescence Switching of Graphene Quantum Dots for Highly Selective Detection of Glutathione

Abstract As a short peptide containing active thiol group, glutathione (GSH) participates in many cellular reactions, so it is of great significance to detect cellular GSH. In this work, amino-rich graphene quantum dots (GQDs) were synthesized, which could coordinate with copper ions (Cu2+) and yield aggregation-induced fluorescence quenching. However, GSH owns stronger coordination ability with Cu2+, so that GSH could promote the dissociation of Cu2+ from the surface of GQDs, and then led to the fluorescence recovery of GQDs. In BR buffer medium at pH 6.8, GSH was able to gradually recover the fluorescence of GQDs (1 μg/mL) that was quenched by Cu2+ (250 μmol/L), which could be finished within 20 min. Additionally, the recovery degree of fluorescence was linear to the concentration of GSH in the range of 20–500 μmol/L with a detection limit of 3.4 μmol/L. This method was applicable to the detection of GSH in cell lysate by the switchable function of Cu2+ to improve the selectivity.

Facile incorporation of DNA-templated quantum dots for sensitive electrochemical detection of the oral cancer biomarker interleukin-8.

Recent studies reveal a great value of interleukin-8 (IL-8), a pro-inflammatory cytokine, as a potent biomarker for early diagnosis of oral cancer. Herein, a new electrochemical method is proposed to detect IL-8 by facilely incorporating DNA-templated quantum dots (QDs). In principle, target IL-8 is first treated with the reducing agent tris(2-carboxyethyl)phosphine (TCEP) to yield active thiols and then captured by antibody-functionalized magnetic beads (MBs). Thereafter, via the Michael addition reaction between the active thiol and maleimide group, a maleimide-modified DNA probe is linked to the surface of MBs, which can initiate a process of rolling circle amplification. In this way, long-range DNA strands are generated on the MB surface, subsequently recruiting DNA-templated CdTe/CdS QDs (DNA-QDs) to act as electrochemical reporters. By tracing the responses of DNA-QDs, the method allows IL-8 detection in a linear range from 5 to 5000fg/mL with a detection limit of 3.36fg/mL. The selectivity, reproducibility, and applicability in complex serum samples are also demonstrated to be favorable, indicating that the method may have a great potential in the future. More importantly, the use of TCEP treatment in the method not only provides a facile way to incorporate DNA-QDs, avoiding the complicated and time-consuming preparation process of antibody-DNA conjugates or functional nanomaterials; but also makes the method capable of being extended to detect other protein biomarkers in view of widespread presence of disulfides, which may hold a broad potential to facilitate efficient biosensing designs.

Characterization of bromelain indicates a molar excess of inhibitor vs. enzyme molecules, a Jacalin-like lectin and Maillard reaction products

The phytotherapeutic bromelain is a heterogeneous protein mixture, extracted from pineapple stem, with high proteolytic activity based on cysteine proteases. Its global protein chemical composition was analyzed qualitatively and quantitatively by SDS-PAGE and RP-HPLC. A SDS-PAGE method with elaborate sample pretreatment was developed, to cope with the bromelain's self-digestion properties and the hypothetical disulfide scrambling during electrophoresis. Both can produce misleading results, if not considered. RP-HPLC was applied for its high separation power for bromelain proteinaceous compounds. A peak identification and assignment to different protein classes in bromelain was done by enzyme kinetics and MS. The method was successfully applied for the quantitative determination of the molar ratio between inhibitor and enzyme and resulted to be approximately 3:2. Bromelain contains, from a molar point of view, inhibitor molecules as major component, which thus might be considered as a natural pharmaceutical excipient in Bromelain, because it protects the enzymes against autolysis. We described two methods to separate the inhibitor fraction from the enzyme fraction, RP-HPLC and size exclusion chromatography. A pineapple derived Jacalin-like-lectin, herein called ‘Anlec’, was identified and quantified by RP-HPLC-MS in bromelain and its content was determined to be 5%, related to all proteins in bromelain. Anlec binds specifically to mannose-containing glycans and is discussed in literature to possess anti-HIV medical potential. Bromelain could therefore be a possible and economic source for the production of Anlec. An isolation strategy of Anlec from bromelain, in high purity, is shown in this work. The presented RP-HPLC results are comprehensive in chemical information, and the method is expedient to provide appropriate bromelain protein isolations but also to accomplish quality control, covering all relevant protein components. It is furthermore shown, that proteins in bromelain may react with reducing sugars in a Maillard reaction to form glycated proteins. Maillard reaction products in bromelain are detected and characterized and could be responsible for the limited stability and storage times at room temperature of bromelain. Even the active center thiol group could be potentially glycated.

Methanol leaf extract of Momordica charantia protects alloxan-induced hepatopathy through modulation of caspase-9 and interleukin-1β signaling pathways in rats

Background and Aim:Momordica charantia is a highly valued plant, widely distributed in the tropical and subtropical regions. The plant is reported to have a wide range of medicinal uses. This study was designed to explore the ameliorative potential of M. charantia methanol leaf extract in alloxan-induced diabetic animal model with a particular focus on the liver.Materials and Methods:Hepatoprotective effect of methanol leaf extract of M. charantia was assessed in alloxan-induced toxicity in 50 rats divided into five groups (A-E) (n=10). Group A normal control, Group B was toxicant group, and Group C animals received glibenclamide treatment while Groups D and E received extracts at 200 and 400 mg/kg doses, respectively. The experiment lasted for 28 days. Histopathological changes, blood glucose level, and serum enzymes such as aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase, oxidative status and caspase-9, and interleukin-1β (IL-1β) were evaluated.Results:Extract-treatment caused a decreased blood glucose level, markers of oxidative stress such as malondialdehyde and hydrogen peroxide (H2O2). Treatment of rats with leaf extract of M. charantia resulted in increased levels and activities of protein thiols, non-protein thiols, glutathione (GSH), glutathione peroxidase, glutathione S-transferase, and superoxide dismutase indicating its antioxidant potential. The liver section revealed mild distortion of the hepatic architecture compared to the toxicant group, while decreased expressions of caspase-9 and IL-1β in extract-treated groups was observed.Conclusion:The plant extract exhibited antioxidant, anti-apoptotic, and anti-inflammatory effects, thus showing its hepatoprotective property.

Oxidants in Physiological Processes.

A number of diseases and conditions have been associated with prolonged or persistent exposure to non-physiological levels of reactive oxygen species (ROS). Similarly, ROS underproduction due to loss-of-function mutations in superoxide or hydrogen peroxide (H2O2)-generating enzymes is a risk factor or causative for certain diseases. However, ROS are required for basic cell functions; in particular the diffusible second messenger H2O2 that serves as signaling molecule in redox processes. This activity sets H2O2 apart from highly reactive oxygen radicals and influences the approach to drug discovery, clinical utility, and therapeutic intervention. Here we review the chemical and biological fundamentals of ROS with emphasis on H2O2 as a signaling conduit and initiator of redox relays and propose an integrated view of physiological versus non-physiological reactive species. Therapeutic interventions that target persistently altered ROS levels should include both selective inhibition of a specific source of primary ROS and careful consideration of a targeted pro-oxidant approach, an avenue that is still underdeveloped. Both strategies require attention to redox dynamics in complex cellular systems, integration of the overall spatiotemporal cellular environment, and target validation to yield effective and safe therapeutics. The only professional primary ROS producers are NADPH oxidases (NOX1-5, DUOX1-2). Many other enzymes, e.g., xanthine oxidase (XO), monoamine oxidases (MAO), lysyl oxidases (LO), lipoxygenase (LOX), and cyclooxygenase (COX), produce superoxide and H2O2 secondary to their primary metabolic function. Superoxide is too reactive to disseminate, but H2O2 is diffusible, only limited by adjacent PRDXs or GPXs, and can be apically secreted and imported into cells through aquaporin (AQP) channels. H2O2 redox signaling includes oxidation of the active site thiol in protein tyrosine phosphatases, which will inhibit their activity and thereby increase tyrosine phosphorylation on target proteins. Essential functions include the oxidative burst by NOX2 as antimicrobial innate immune response; gastrointestinal NOX1 and DUOX2 generating low H2O2 concentrations sufficient to trigger antivirulence mechanisms; and thyroidal DUOX2 essential for providing H2O2 reduced by TPO to oxidize iodide to an iodinating form which is then attached to tyrosyls in TG. Loss-of-function (LoF) variants in TPO or DUOX2 cause congenital hypothyroidism and LoF variants in the NOX2 complex chronic granulomatous disease.

A Review of Cannabis and Interactions With Anticoagulant and Antiplatelet Agents

Legalization of medical cannabis has occurred in 33 states and the District of Columbia, and recreational use has increased exponentially since 2013. As a result, it is important to understand how cannabis interacts with other drugs and has potential risks for patients on concomitant medications. Components of medical cannabis can inhibit or compete for several cytochrome P450 (CYP) hepatic isoenzymes, UDP-glucuronosyltransferases, and P-glycoprotein. These enzymes and transporters are involved in the metabolism and absorption of numerous medications, including anticoagulants (ACs) and antiplatelet agents (APs), potentially causing harmful drug-drug interactions. ACs and/or APs are often prescribed to high-risk patients with cardiac conditions, a history of myocardial infarction, or stroke. Cannabis may cause these medications to be less efficacious and put patients at risk for recurrent cardiovascular and cerebrovascular events. Several case reports show cannabis may inhibit the metabolism of warfarin because of CYP2C9 interactions, resulting in increased plasma concentrations, increased international normalized ratio, and risk of bleeding. Cannabidiol inhibits CYP2C19, an isoenzyme responsible for the transformation of clopidogrel to its active thiol metabolite. This interaction could lead to subtherapeutic levels of active metabolite and possibly increased stroke risk. Within this review, a total of 665 articles were screened from PubMed and EMBASE. Four case reports, 1 in vitro study, and 1 pharmacokinetic article were found to be of relevance. This review serves to examine reported and potential cannabis interactions with APs/ACs to help inform patients and health care providers of possible risks and knowledge gaps.

Development and validation of a UPLC–MS/MS method for simultaneous determination of vicagrel and its major metabolites in rat or human plasma: An optimized novel strategy for the stabilization of vicagrel

Vicagrel is a promising novel antiplatelet drug. However, the quantification of vicagrel in plasma is currently unavailable since it is liable to be hydrolyzed in plasma by esterases. In this study, an optimized strategy was developed and validated to stabilize vicagrel, 2-oxo-clopidogrel (thiolactone metabolite), and H4 (active thiol metabolite) before quantification of the analytes, such as addition of citric acid (for plasma acidification) and NaF (a non-specific esterase inhibitor) to inhibit esterase activity, immediate addition of a thiol-alkylating reagent MPB into blood samples to derivatize H4 for the formation of stable H4 derivative (i.e., MP-H4), use of the anticoagulant K2EDTA to minimize the conversion of 2-oxo-clopidogrel to H-endo, and keeping the analytes at 4 °C or on wet ice to minimize degradation of the analytes when processed and analyzed. The stability was measured as percent of each analyte remained in plasma samples after their storage for 4 h at 4 °C or in blood samples after 1 h at 4 °C. The results indicated that stability of vicagrel was increased significantly in stabilized plasma or blood samples compared with non-stabilized controls for rats and humans, respectively, and that the stability of 2-oxo-clopidogrel was increased to a certain extent. In contrast, MP-H4 formed was stable in plasma immediately after thorough mixture of MPB with blood. We conclude that the above strategy is useful for improving the stability of vicagrel, 2-oxo-clopidogrel, and H4 in rat or human plasma, and that vicagrel and its two major metabolites can be quantified accurately and simultaneously.

High-performance sono/nano-catalytic system: Fe3O4@Pd/CaCO3-DTT core/shell nanostructures, a suitable alternative for traditional reducing agents for antibodies

Herein, a novel heterogeneous nanoscale reducing agent for antibody cleavage, made of iron oxide nanoparticles, silica network, palladium on calcium carbonate (10%), and dithiothreitol (Fe3O4@Pd/CaCO3-DTT), is presented as a substantial alternative for traditional homogeneous analogues. Conventionally, antibody fragmentation is accomplished using reducing agents and proteases that digest or cleave certain portions of the immunoglobulin protein structure to provide active thiol sites for drug tagging aims. Then, dialysis process is needed to separate excess chemical structures and purify the reduced antibody. In this work, we have made an effort to design a suitable heterogeneous tool for protein cleavage and skip the dialysis process for purification of the reduced antibody. In this regard, firstly, various preparation methods including microwave irradiation, reflux and ultrasonication have been precisely compared, and it has been proven that the best result is obtained through 10 min ultrasound (US) irradiation using an US bath with 50 KHz frequency and 200 W L-1 power density. Then, all the necessary structural analyses have been done and thoroughly interpreted for the final product. Afterward, the catalytic performance of Fe3O4@Pd/CaCO3-DTT nanoscale system in the presence of US waves (50 KHz, 200 W) has been monitored using some disulphide derivatives. The NPs could be conveniently separated from the mixture through their substantial paramagnetic property. Thus, dialysis process in which various types of membranes are used is practically jumped after the reduction process. In this work, this is clearly demonstrated that there is a constructive synergistic effect between US waves and prepared Fe3O4@Pd/CaCO3-DTT nanoscale reducing agent. Ultimately, trastuzumab (anti HER-2) antibody has been used to test the performance of the prepared Fe3O4@Pd/CaCO3-DTT NPs in a real protein reduction reaction.

Bioinspired poly (γ-glutamic acid) hydrogels for enhanced chondrogenesis of bone marrow-derived mesenchymal stem cells

While peptide-directed scaffolds now serve as well-established platforms for biomimetic three-dimensional (3D) extracellular matrices (ECM), challenges still remain for chondrogenesis through direct mediation of stem cells. Here, biocompatible poly (γ-glutamic acid) (γ-PGA) hydrogels with robust mechanical properties were developed based on methacrylate-γ-PGA (γ-PGA-GMA) and cysteamine functionalized γ-PGA (γ-PGA-SH) for cartilage regeneration. The γ-PGA hydrogels demonstrated good self-crosslinking property as well as tunability through conjugation between active thiol groups of γ-PGA-SH and methacrylate moieties of γ-PGA-GMA. The mechanical property, porous structure, swelling, and degradation process of the hydrogels could be controlled by adjusting modified γ-PGA polymers component. The rheological behavior and compression test of γ-PGA hydrogels illustrated a wide processing range in addition to superb mechanical properties. These γ-PGA hydrogels showed excellent elasticity as well as toughness, withstanding more than 70% of mechanical strain. Meanwhile, the stress modulus of γ-PGA hydrogels could be up to 749 kPa. We also studied γ-PGA hydrogels as scaffolds for the 3D culture and chondrogenesis differentiation of rabbit bone marrow-derived mesenchymal stem cells (BMSCs) in vitro. In a rabbit auricular cartilage defect model, BMSCs-laden hydrogel effectively promoted chondrogenesis. Based on these findings, biomimetic γ-PGA-based hydrogels hold promising application as favorable scaffold biomaterials for cartilage tissue regeneration.

Glutathione-glutaredoxin is an efficient electron donor system for mammalian p53R2–R1-dependent ribonucleotide reductase

Deoxyribonucleotides are DNA building blocks and are produced de novo by reduction of ribose to deoxyribose. This reduction is catalyzed by ribonucleotide reductase (RNR), a heterodimeric tetramer enzyme in mammalian cells, having one of two free radical-containing subunits called R2 and p53R2. R2 is S-phase specific and used for DNA replication, whereas p53R2 functions in DNA repair and mitochondrial DNA synthesis. The larger RNR subunit, R1, has catalytically active cysteine thiols in its buried active site and a C-terminal swinging arm, with a Cys-Leu-Met-Cys sequence suggested to act as a shuttle dithiol/disulfide for electron transport. After each catalytic cycle the active site contains a disulfide, which has to be reduced for turnover. Thioredoxin (Trx) and glutaredoxin (Grx) systems have been implicated as electron donors for the RNR disulfide reduction via the swinging arm. Using mouse R1-R2 and R1-p53R2 complexes, we found here that the catalytic efficiency of the GSH-Grx system is 4-6 times higher than that of the Trx1 system. For both complexes, the Vmax values for Grx are strongly depended on GSH concentrations. The GSH disulfide resulting from the Grx reaction was reduced by NADPH and GSH reductase and this enzyme was essential because reaction with GSH alone yielded only little activity. These results indicate that C-terminal shuttle dithiols of mammalian R1 have a crucial catalytic role and that the GSH-Grx system favors the R1-p53R2 enzyme for DNA replication in hypoxic conditions, mitochondrial DNA synthesis, and in DNA repair outside the S-phase.

Preventing Radiation-Induced Injury by Topical Application of an Amifostine Metabolite-Loaded Thermogel

Despite the development of high-precision radiation therapy, ionizing radiation inevitably damages healthy tissues. Radiodermatitis and radioinduced oral mucositis are frequent and significant side effects among patients with breast and head and neck cancer, respectively. These radiation-related injuries negatively affect patient quality of life and can lead to unplanned therapeutic breaks and compromise treatment outcomes. Currently, no preventive or mitigating agent has emerged to address these issues. Although amifostine, a well-known free radical scavenger, has proven efficacy against specific radio- and chemo-induced toxicities, severe adverse side effects (reversible hypotension, nausea, emesis, etc) combined with logistical hurdles are associated with its recommended intravenous route of administration, limiting its use. We developed a thermogel containing the active thiol metabolite of amifostine (CPh-1014) that polymerizes at body temperature and serves as a matrix for topical application onto the skin or mucosa. Applied before irradiation, CPh-1014 greatly reduced the severity of oral mucositis and dermatitis induced byeither a single dose or fractionated irradiation regimens in invivo mouse models. The cytoprotective effect of CPh-1014 was confirmed by the decrease in DNA double-strand breaks in the irradiated epithelium. Noticeably, CPh-1014 did not affect radiation therapy efficacy against tumors grafted at submucosal and subcutaneous sites. Incontrast to the intravenous administration of amifostine, CPh-1014 oral application did not induce hypotension indogs. CPh-1014 confers radioprotective effects in healthy tissues with reduced systemic side effects without compromising radiation therapy efficacy. We propose CPh-1014 as an easy-to-implement therapeutic approach to alleviate radiation therapy toxicity in patients with breast and head and neck cancer.

Site-specific protein modification by genetic encoded disulfide compatible thiols

Cysteine chemistry provides a low cost and convenient way for site-specific protein modification. However, recombinant expression of disulfide bonding containing protein with unpaired cysteine is technically challenging and the resulting protein often suffers from significantly reduced yield and activity. Here we used genetic code expansion technique to introduce a surface exposed self-paired di-thiol functional group into proteins, which can be selectively reduced to afford active thiols. Two compounds containing self-paired disulfides were synthesized, and their genetic incorporations were validated using green fluorescent proteins (GFP). The compatibility of these self-paired di-thiols with natural disulfide bond was demonstrated using antibody fragment to afford site-specifically labeled antibody. This work provides another valuable building block into the chemical tool-box for site-specific labeling of proteins containing internal disulfides.

Redox Activation of Hydrogen Sulfide, Thiols, and Sulfur in Electrosynthesis of Organic Di- and Polysulfides

A novel and efficient method for the synthesis of biologically active organic di-, tri- and tetrasulfides has been proposed. Different methods of redox activation of sulfur, hydrogen sulfide, and thiols in the reactions with organic compounds have been considered. Electrochemical initiation of the reactions of the mediator–H2S–S8 system with cyclohexane, methylcyclohexane, and benzene has occurred to the formation of polysulfides R2Sn (n = 2–4). The application of tetrabutylammonium bromide as a mediator of H2S oxidation has allowed to decrease the anodic overpotential of electrosynthesis. Di- and tetrasulfides have been obtained under anodic activation of the cycloalkanethiols (C5, C6) or thiophenol in the reaction with sulfur. Electroreduction of S8 in the presence of the same thiols has favored the formation of di- and trisulfides. The yield and the ratio of the R5Sn (n = 2–4) depend on the method of redox activation of the thiolating reagent.

Lewis Acid Promoted Aerobic Oxidative Coupling of Thiols with Phosphonates by Simple Nickel(II) Catalyst: Substrate Scope and Mechanistic Studies

Exploring new catalysts for efficient organic synthesis is among the most attractive topics in chemistry. Here, using Ni(OAc)2/LA as catalyst (LA: Lewis acid), a novel catalyst strategy was developed for oxidative coupling of thiols and phosphonates to phosphorothioates with oxygen oxidant. The present study discloses that when Ni(OAc)2 alone was employed as the catalyst, the reaction proceeded very sluggishly with low yield, whereas adding non-redox-active metal ions such as Y3+ to Ni(OAc)2 dramatically promoted its catalytic efficiency. The promotional effect is highly Lewis acidity dependent on the added Lewis acid, and generally, a stronger Lewis acid provided a better promotional effect. The stopped-flow kinetics confirmed that adding Y(OTf)3 can obviously accelerate the activation of thiols by Ni(II) and next accelerate its reaction with phosphonate to generate the phosphorothioate product. ESI-MS characterizations of the catalyst disclosed the formation of the heterobimetallic Ni(II)/Y(III) species in the catalyst solution. Additionally, this Ni(II)/LA catalyst can be applied in the synthesis of a series of phosphorothioate compounds including several commercial bioactive compounds. This catalyst strategy has clearly supported that Lewis acid can significantly improve the catalytic efficiency of these traditional metal ions in organic synthesis, thus opening up new opportunities in their catalyst design.

Thiol-functionalized magnetic nanoparticles for static and dynamic removal of Pb(II) ions from waters

The synthesis of magnetite (Fe3O4) nanoparticles (NPs) and the functionalization by means of a silane derivative (3-mercaptopropyl) trimethoxysilane (MPTMS) as a way for heavy metal capture, i.e., Pb2+ ions, has been investigated. A direct reaction between the active thiol organosilane molecule and the surface of the magnetite nanoparticle is described. The coated nanoparticles, presenting an average diameter of about 20 nm, have been extensively characterized from the morphological, chemical, and physical point of view. The capture of Pb2+ ions from aqueous solution has been performed both in static and dynamic mode, using in both cases a configuration of commercial NdFeB permanent magnets. The amount of heavy metal captured by the magnetic NPs as a function of time, as well as its fraction recovered by an EDTA solution, have been measured by inductively coupled plasma atomic emission spectrometry (ICP-AES). The Pb2+ capture from a spiked water solution (5 mg/L) is very fast, reaching 91% in static conditions after only 10 min. Furthermore, dynamic data also demonstrated the possibility to apply the toxic heavy metal capture method to large volumes.

A robust MOF-based trap with high-density active alkyl thiol for the super-efficient capture of mercury.

The first Zr-based metal-organic frameworks (MOFs) with high-density active alkyl thiol were successfully constructed in an aqueous phase. Thanks to the strong affinity between alkyl thiol and Hg2+ ions, the robust MOFs feature high adsorption capacity (734 mg g-1), ultra-fast kinetics (removing 99.99% Hg2+ in 5 min), super-high capture efficiency (dropping Hg2+ concentration from 10 000 ppb to 0.11 ppb), wide working range of pH, high selectivity and good reusability.

Using Flow Cytometry to Detect and Measure Intracellular Thiol Redox Status in Viable T Cells from Heterogeneous Populations.

Increased production of reactive oxygen species (ROS) and deficiencies in cellular antioxidant defenses are the principal causes of cellular oxidative stress. ROS can react with a variety intracellular molecules, including redox active cysteine thiols (-SH) within proteins. Cysteine thiols can occupy several redox states and conversion between them is highly dynamic during, for example, cell growth, resulting in modification and subsequent loss of the "reduced thiol" form (-SH or -S-). The challenge lies with detecting and measuring thiol redox status inside viable heterogeneous cell populations (e.g., peripheral blood mononuclear cells (PBMCs)). Here we describe a flow cytometric approach for the evaluation of intracellular thiol redox status in human CD3+ T cells within a viable PBMC preparation. Using the thiol reactive probe, fluorescein-5 maleimide (F5M), we demonstrate that loss of reduced intracellular thiol correlates with a decrease in F5M fluorescence. We also detected a loss of F5M fluorescence in Jurkat cell cultures exposed to exogenous H2O2 generated by glucose oxidase. Since F5M binds irreversibly to reduced cysteine thiols, cells may be sorted based on F5M fluorescence intensity and redox active proteins can subsequently be extracted and separated using SDS-PAGE. This final step facilitates identification of redox active proteins from individual cell populations in live heterogeneous cell mixes using proteomic analysis.

Redox-Regulated, Targeted Affinity Isolation of NADH-Dependent Protein Interactions with the Branched Chain Aminotransferase Proteins.

Isolation and identification of protein targets for redox-active proteins is challenging. The human branched chain aminotransferase (hBCAT) proteins are redox active transaminases that can be regulated through oxidation, S-nitrosation and S-glutathionylation. This metabolic protein was shown to associate with the E1 decarboxylase component of the branched-chain α-keto acid dehydrogenase complex in a NADH-dependent manner, where mutation of the CXXC center was shown to prevent complex formation. To determine if the redox state of the CXXC motif can influence other NADH-dependent protein-protein interactions, proteins were extracted from neuronal cells treated under reduced and oxidized conditions and then isolated using targeted affinity chromatography, resolved using 2D electrophoresis. Select proteins spots were excised and identified using a quadrupole time of flight mass spectrometer (Thermo) with a precursor tolerance of 10ppm and subsequently analyzed using Proteome Discoverer 2.1 with Swiss-Prot human DB. Mass tolerances for precursor/product were set to 10ppm/0.6Da and data were filtered by peptide confidence with PD2.1. It was determined that the protein profile considerably altered in both number and abundance dependent on the redox state of the cell and also on the availability of the redox active thiol groups. The biological relevance of the newly identified partners was determined using DAVID, the bioinformatics database, which indicated that proteins important to cytoskeletal function, protein transport, protein synthesis, chaperone activity, and cell signaling.

Reduction of molecular oxygen by redox active thiols: comparison of glutathione, N-acetylcysteine, cysteine, and homocysteine.

The reaction properties of the thiol compounds, cysteine (Cys), N-acetyl-l-cysteine (NAC), the reduced form glutathione (GSH), and homocysteine (HCS) were compared. The main purpose of this study was to find a thiol-based anti-oxidant suitable for biological experiments and to provide clear reasoning for its selection. The availability of thiol compounds to generate superoxide by reducing molecular oxygen (O2) at a hyperthermal temperature was discussed. An oxidative atmosphere, i.e., superoxide generation by the hypoxanthine-xanthine oxidase reaction, hydroxyl radical generation by X-ray irradiation, or direct one-electron oxidation by ferricyanide, was prepared in a reaction mixture containing 0.1 mM TEMPOL and 1 mM test compound, and the EPR signal decay of TEMPOL was observed. A reaction mixture containing 0.1 mM TEMPOL and 1 mM thiol compound was incubated at 44°C, and the EPR signal decay of TEMPOL was observed. Thiols could function as H-donors to the oxoammonium cation and produce the hydroxylamine form of TEMPOL in an oxidative atmosphere. Thiols could also irreversibly react with the oxoammonium cation. GSH and Cys could reduce O2 to form superoxide/hydroperoxyl radical at hyperthermal temperatures, but HCS and NAC could not reduce O2. GSH and Cys may cause reductive stress, whereas NAC is a simple tractable antioxidant.